Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood © 2022 the Authors Accepted version (Final draft) Kolehmainen, Pekka; Heroum, Jemna; Jalkanen, Pinja; Huttunen, Moona; Toivonen, Laura; Marjomäki, Varpu; Waris, Matti; Smura, Teemu; Kakkola, Laura; Tauriainen, Sisko; Peltola, Ville; Julkunen, Ilkka Kolehmainen, P., Heroum, J., Jalkanen, P., Huttunen, M., Toivonen, L., Marjomäki, V., Waris, M., Smura, T., Kakkola, L., Tauriainen, S., Peltola, V., & Julkunen, I. (2022). Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood. Microbiology Spectrum, 10(3), e01967-21. https://doi.org/10.1128/spectrum.01967-21 2022 Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood Pekka Kolehmainen, a Jemna Heroum, a Pinja Jalkanen, a Moona Huttunen, a Laura Toivonen, b Varpu Marjomäki, c Matti Waris, a , e Teemu Smura, d Laura Kakkola, a Sisko Tauriainen, a Ville Peltola, b Ilkka Julkunen a , e a Institute of Biomedicine, University of Turku, Turku, Finland b Department of Paediatrics and Adolescent Medicine, Turku University Hospital and University of Turku, Turku, Finland c Department of Biological and Environmental Sciences/Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland d Department of Virology, University of Helsinki, Helsinki, Finland e Clinical Microbiology, Turku University Hospital, Turku, Finland ABSTRACT Seasonal human coronaviruses (HCoVs) cause respiratory infections, especially in children. Currently, the knowledge on early childhood seasonal coronavirus infections and the duration of antibody levels following the first infections is limited. Here we analyzed serological follow-up samples to estimate the rate of primary infection and reinfection(s) caused by seasonal coronaviruses in early childhood. Serum specimens were collected from 140 children at ages of 13, 24, and 36 months (1, 2, and 3 years), and IgG antibody levels against recombinant HCoV nucleoproteins (N) were measured by enzyme immunoassay (EIA). Altogether, 84% (118/140) of the children were seropositive for at least one seasonal coronavirus N by the age of 3 years. Cumulative seroprevalences for HCoVs 229E, HKU1, NL63, and OC43 increased by age, and they were 45%, 27%, 70%, and 44%, respectively, at the age of 3 years. Increased antibody levels between yearly samples indicated reinfections by 229E, NL63, and OC43 viruses in 20– 48% of previously seropositive children by the age of 3 years. Antibody levels declined 54–73% or 31–77% during the year after seropositivity in children initially seropositive at 1 or 2 years of age, respectively, in case there was no reinfection. The correlation of 229E and NL63, and OC43 and HKU1 EIA results, suggested potential cross-reactivity between the N specific antibodies inside the coronavirus genera. The data shows that seasonal coronavirus infections and reinfections are common in early childhood and the antibody levels decline relatively rapidly. IMPORTANCE The rapid spread of COVID-19 requires better knowledge on the rate of coronavirus infections and coronavirus specific antibody responses in different population groups. In this work we analyzed changes in seasonal human coronavirus specific antibodies in young children participating in a prospective 3-year serological follow-up study. We show that based on seropositivity and changes in serum coronavirus antibody levels, coronavirus infections and reinfections are common in early childhood and the antibodies elicited by the infection decline relatively rapidly. These observations provide further information on the characteristics of humoral immune responses of coronavirus infections in children. KEYWORDS 229E, HKU1, NL63, OC43, seasonal coronavirus, serology, respiratory infection, antibodies, enzyme immunoassay, children The family of coronaviruses consists of enveloped positive-strand RNA viruses, which are further classified into four genera, alpha-, beta-, gammaand deltacoronaviruses, based on their phylogenetic relationships (1). Human coronaviruses (HCoVs) can cause respiratory infections ranging from subclinical and mild infections to a severe acute respiratory syndrome (SARS) (2). Four of the HCoVs, “seasonal”HCoVs, are low Editor Takamasa Ueno, Kumamoto University Ad Hoc Peer Reviewer Koji Watanabe, National Center For Global Health and Medicine Copyright © 2022 Kolehmainen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Pekka Kolehmainen, pekka.j.kolehmainen@utu.fi. The authors declare no conflict of interest. Received 30 October 2021 Accepted 17 February 2022 Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 1 RESEARCH ARTICLE Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. pathogenic, endemic viruses and their infections are common in all age groups, especially in children under 10 years of age (3). Seasonal HCoVs may be detected in respiratory tract samples from hospitalized children although they are rarely the causative agent of a severe respiratory infection (4). Seroconversion for the seasonal alphacoronaviruses has been estimated to occur on an average at the age of 3.5 years (5), and seroprevalence reaches its maximum at the age of 5 years (6). Genetically and biologically HCoVs are distinct from each other. All HCoVs can cause respiratory infections (7) although they have differential preferences for host cell tropism and receptor molecules (7, 8). Two of the seasonal HCoV species, 229E and NL63, are members of the genus alphacoronavirus while HKU-1 and OC43 belong to genus betacoronavirus, which also includes Middle East respiratory syndrome virus (MERS), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Obtaining accurate data on the prevalence of seasonal coronavirus infections is challenging since reinfections are common and antibody levels may wane relatively rapidly (9, 10). However, antibody levels remain detectable for up to 2 years (9, 11), which supports the use of serological assays for estimating coronavirus infection burden in early childhood. This study increases our knowledge on the rate of seasonal HCoV infections in early childhood. We used nucleoprotein-based (N) enzyme immunoassay (EIA) to analyze IgG antibody levels against four seasonal coronaviruses in 140 children in Finland with sequential serum samples collected at the ages of 1, 2, and 3 years in a setting of a prospective follow-up birth cohort. RESULTS Seasonal HCoV seroprevalence in children. To study the seropositivity and IgG antibody levels for seasonal HCoVs in young children, we analyzed sequential serum samples collected at the age of 1, 2 and 3 years from 140 children for HCoV N-proteinspecific IgG antibodies using EIA. The analysis showed differences in the prevalence of elevated antibody levels between HCoVs (Fig. 1A). In all of the age groups the antibody positivity rate was highest for NL63 (21% in 1-year-old, 31% in 2-year-old, and 57% in 3-year-old children) followed by 229E (16, 27, and 37%), OC43 (14, 25, and 29%), and HKU1 (6, 16, and 14%) (Fig. 1B). The seropositivity increased with age, except for HKU1, which had a lower seropositivity in 3-year-old children in comparison to 2-year-old children. The cumulative seropositivityat2and3yearsofagewashighercomparedtotheyearlyseropositivity.Thisdifference resulted from children turning seronegative after the earlier seropositivity. The proportion of children who were seronegative for 229E, HKU1, NL63, and OC43 at 2 years after seropositivity at 1 year was 32% (n= 7/22), 63% (n= 5/8), 48% (n= 14/29), and 47% (n= 9/19). Of children who were seropositive for 229E, HKU1, NL63, and OC43 at 2 years, 18% (n=7/ 38), 64% (n= 14/22), 30% (13/44), and 40% (14/35) were seronegative at the age of 3 years. Inthe3-year-oldchildren,thecumulativeseropositivitywashighestforNL63(70%),followed by 229E (45%), OC43 (44%), and HKU1 (27%). Altogether, 45% (63/140) of the 1-year-old children had anti-N IgG antibodies against at least one of the seasonal coronaviruses indicating that these children had experienced at least one HCoV infection by the age of 1 year (Fig. 1B). The proportion of children with antibodies for at least one seasonal coronavirus increased to 67% by 2 years and to 84% by 3 years of age. Sixteen percent of the children had no antibodies for any seasonal coronaviruses during the follow-up. Of all 420 serum samples, eight (1.9%) and nine (2.1%) showed IgG antibody levels above the cutoff for MERS and SARS-CoV-2 N proteins, respectively. Antibody levels induced by seasonal HCoV infection and reinfection. To estimate the changes in IgG antibody levels following primary infection, the children were grouped based on the age they were seropositive for the first time. In children who were seropositive for any of the seasonal coronaviruses at the age of 1 year, the geometric mean IgG antibody levels (GMALs) decreased by the age of 2 years (229E 30 to 16 EIA units, P= 0.029; HKU1 23 to 10, ns; NL63 33 to 11, P= 0.004; OC43 23 to 16, ns; Fig. 2A). In these same children, the GMALs continued to decrease for HKU1 and OC43 Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 2 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. from 2 to 3 years (10 to 5 EIA units, ns; 16 to 11, ns, respectively), whereas for 229E and NL63 the antibody levels between individuals varied greatly at 3 years, which resulted in slightly increased GMALs (16 to 20 EIA units, ns; 11 to 22, P= 0.041, respectively). Similar trends were observed in children who experienced their primary infection between 1 and 2 years (Fig. 2B): GMALs for HKU1 and OC43 decreased significantly (29 to 7 EIA units, P,0.0001; 32 to 12, P= 0.008) from 2 to 3 years, whereas for 229E and NL63 the antibody levels between individuals showed great variation and no significant changes were observed in GMALs from 2 to 3 years (24 to 25 EIA units, ns; 25 to 21, ns, respectively). At the age of 3 years, the children who turned seropositive for FIG 1 Seropositivity and IgG antibody responses for six HCoV N proteins in children. Anti-N IgG antibody levels were measured with EIA. Serum samples were collected from 140 children at the age of 1, 2, and 3 years and analyzed for IgG antibodies against 6 HCoV N proteins (A). Cutoff value is indicated with a dashed line. Antibody levels are shown as EIA units, which have been calculated in relation to positive (100 EIA units) and negative (0 EIA units) control specimen pools. Samples with an EIA unit value lower than 1 were given a value 1 enabling the calculation of geometric means. The rates of N IgG seropositivity for seasonal HCoVs, and for any seasonal HCoV in different age groups (shown as bars) and as a cumulative seropositivity (shown as triangles) are shown (B). Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 3 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. seasonal HCoVs between the age of 2 and 3 years, had higher GMALs than the children who had their primary infection earlier (Fig. 2A to C). IgG antibody levels varied greatly between individuals after primary infection: some of the children turned clearly seronegative and others had significant increases in their antibody levels. This variation indicates either a decline in antibody levels in the absence of a reinfection or an increase in antibody levels due to a reinfection between sample collections. Serum samples from children who were seropositive for a seasonal HCoV at 1 or 2 years were further analyzed for reinfection, which was defined as an increase of $20 EIA units in comparison to the previous serum sample. Significantly higher GMALs were observed in FIG 2 IgG antibody levels in children following and preceding the primary infection. The children were separated into different groups based on the time they turned seropositive: before the age of 1 year (A), between 1 and 2 years (B), and between 2 and 3 years (C). The number of children in each group is presented in the graphs. Lines connect the IgG levels of an individual in each time point. Orange lines indicate individuals who have an increase of .20 EIA units in the antibody levels after initial seropositivity. Cutoff values are indicated as dashed lines. Statistical differences in IgG levels between different age groups were analyzed using Wilcoxon matched pairs signed-rank test. Two-tailed P-values *,0.05, **,0.01, ***,0.001, and ****,0.0001 were considered significant. Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 4 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. children who had an indication of a reinfection although some groups of children were too small for statistical analysis (Fig. S1 in the supplemental material). The antibody levels of 1-year-old seropositive children increased following a reinfection between 1 and 2 years but decreased by the age of 3 years. In children who were seropositive for an HCoV by 2 years and showed no indication of a reinfection, the GMALs decreased to low levels (816 EIA units) by the age of 3 years (Fig. S1 and S2). To analyze the kinetics of the decline of anti-HCoV IgG antibodies, the seropositive children with no indication of reinfection were divided into two groups based on the age they had turned seropositive. Following seropositivity at the age of 1 year, the GMALs for any HCoV decreased by 54–73% until the age of 2 years (35 to 12 EIA units for 229E, P= 0.0017; 23 to 11 for HKU1, ns; 32 to 9 for NL63, P= 0.005; 23 to 10 for OC43, P= 0.011, respectively; Fig. S2A in the supplemental material). The following year GMALs remained low in all of these groups. A similar decline in GMALs (70–77%) was seen from 2 to 3 years in children with a primary seropositivity at 2 years of age for HKU1 (29 to 7 EIA units, P= 0.0003), NL63 (31 to 9, P= 0.0007) and OC43 (34 to 10, P= 0.0001; Fig. S2B). For 229E, GMAL of children with a primary seropositivity at 2 years of age decreased by 31% (23 to 16 EIA units, ns), which is less than the decrease in antibody levels for other HCoVs. This reduced decrease in the GMAL was affected by several samples which, instead of a decrease, showed a weak (,20 EIA units) antibody increase (Fig. S1), potentially due to a reinfection that failed to increase IgG antibody levels over the reinfection cutoff or cross-reactive antibodies. Correlation of anti-N antibody levels between different virus types. To evaluate potential cross-reactivity between anti-N protein antibodies, we calculated the correlation coefficients between EIA data for all serum samples (n= 420). The levels of IgG antibodies against 229E correlated moderately with the levels of anti-NL63 antibodies (r= 0.4388; Fig. 3). The correlation between the levels of anti-OC43 and anti-HKU1 antibodies was relatively strong (r= 0.7003), while the correlation rates of other anti-N protein antibody pairs were low (r,0.18). If samples that were negative for both of the assays were removed from the calculation, the correlation between anti-OC43 and anti-HKU1 antibodies remained relatively strong (Fig. S3 in the supplemental material). However, the correlation between anti-229E and anti-NL63 antibodies weakened drastically, suggesting that negative samples may overemphasize the correlations between the assay pairs. The greater identities in N amino acid sequence of 229E and NL63 (48%) and of OC43 and HKU1 (66%) in comparison with their identity to other HCoVs (22-32%; Fig. S4) may contribute to better correlation coefficients. Validation of 229E and OC43 EIA with IFA. To validate the EIA results we set up an IFA using 229E and OC43 virus-infected Huh7 cells (Fig. 4A). We analyzed 30 EIA-positive and 30 EIA-negative (randomly selected) serum samples to estimate the presence of whole-virus-specific antibodies and to determine IF antibody titer. The concordance between negative and positive samples in 229E IFA and EIA was 83% (20/30 of 229E EIApositive and 0/30 of the 229E EIA-negative samples were positive in 229E IFA; Fig. 4B). Between OC43 IFA and EIA the concordance was also 83% (30/30 of OC43 EIA-positive and 10/30 OC43 EIA-negative samples were OC43 IFA-positive). Higher IFA titers were detected for EIA-positive samples in OC43 IFA than in 229E IFA (geometric mean titer 182 for OC43 and 37 for 229E) although in EIA the corresponding samples had lower geometric mean antibody levels for OC43 than for 229E (31 and 42 EIA units). The IFA titers for 229E and OC43 specific antibodies correlated well with the corresponding EIA unit values (229E EIA and IFA r= 0.7258, and OC43 EIA and IFA r= 0.7809; Fig. 4C). Early childhood seropositivity and reinfections for seasonal HCoVs. IgG antibodies against 229E, HKU1, NL63 and OC43 N-proteins were detected in 16% (23/140), 6% (8/ 140), 21% (30/140) and 14% (20/140) of children by the age of 1 year (Fig. 5). During the follow up, the number of seropositive children increased: the ratio of children who turned seropositive between 1 and 2 years and 2 and 3 years were 16% and 13% for 229E, 14% and 8% for HKU1, 19% and 30% for NL63, and 18% and 12% for OC43, respectively. Increases in antibody levels indicated that 36% (16/45) of the 229E-seropositive children had two or three 229E-infections by the age of 3 years. For HKU1, NL63 and OC43 Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 5 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. the corresponding reinfection rates were 0% (0/38), 48% (28/58) and 20% (9/44), respectively. One child showed an increase in 229E specific IgG antibody levels between 1 and 2 years of age as well as between 2 and 3 years of age indicating two reinfections. A summary of the numbers and percentages of infections and reinfections in different age groups is presented in Fig. 5. DISCUSSION In this study, we estimated seasonal HCoV (229E, HKU1, OC43, and NL63) infection and reinfection rates by analyzing the presence of anti-coronavirus antibodies in serially collected serum specimens from 140 children between 1 and 3 years of age. Full-length N was selected as antigen for the EIA because it (i) is conserved within coronavirus species, (ii) is highly immunogenic and abundantly expressed, (iii) is relatively different from one coronavirus species to another, and (iv) includes all N epitopes. Previously, the C-terminal partoftheNwasusedasamorespecific antigen to avoid cross-reactivity between coronavirus species (5, 12). However, a recent work including study participants with sequential samples suggested that an assay with the C-terminal part of N as an antigen may also detect antibodies elicited against other seasonal coronavirus species (10), which promoted the selection of full-length N proteins as antigens for this study. Previous studies have reported observations of cross-reactivity in serological responses to HCoVs (13, 14). The higher correlation coefficient rates between HKU1 and OC43 or 229E FIG 3 Correlation of anti-HCoV N IgG antibody levels between different HCoV types. Correlation coefficients of IgG antibody levels for anti-HCoV N assays were determined with Pearson's correlation test. Calculations were done for all samples (n= 420) including negative EIA unit values that were given an arbitrary value of 1. Correlation coefficients (r), Pvalues and linear regression lines are shown. Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 6 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. and NL63 antibodies in our data indicated possible existence of cross-reactive antibodies recognizing N antigens of the same coronavirus genus. However, the knowledge on detailed infection history from each individual would be required to confirm the presence of crossreactivity. To confirmtheresultsfromN-basedEIA,weusedIFAwithvirus-infectedcellsto detect 229E or OC43 virus-specific antibodies in the sera. The comparison of 229E IFA and EIA results indicates that for the detection of anti-229E IgG antibodies EIA is somewhat more sensitive than IFA. This observation was further apparent in the relatively high EIA unit levels in comparison to IFA titers. On the contrary, the IFA results with OC43 virus-infected cells suggested that OC43 anti-N protein IgG EIA may miss some OC43 antibody positive sera. It maybethat229ENisrelativelymoreimmunogenicincomparisontoOC43Nproteinor that the folding of the OC43 N protein in IFA versus EIA affects the binding of the antibodies. Despite a good correlation of EIA and IFA for 229E and OC43, the lack of IFA for HKU1 and especially for NL63 limits the IFA-based validation of EIA in this study. Unlike some recent FIG 4 An example of a dilution series of an IgG positive serum for OC43 and a negative serum sample (A). Permeabilized virus-infected cells were incubated with serum dilutions and with rabbit anti-OC43-N, followed by secondary anti-human (green) and anti-rabbit (magenta) antibodies. Cell nuclei were labeled with DAPI. IgG antibody titers of 60 serum samples detected with OC43 or 229E virus-infected Huh-7 cell line based IFA (B). Correlation between IgG levels in EIA and antibody titers in IFA for 229E-EIA and 229E-IFA as well as OC43-EIA and OC43-IFA was analyzed (C). Spearman’s ranked correlation test coefficients (r), Pvalues and regression lines are shown. (D) IgG antibodies detected for OC43 and 229E with indirect immunofluorescence assay (IFA). Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 7 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249. studies (15–17), we did not detect cross-reactivity between anti-SARS-CoV-2 and anti-seasonal HCoV N protein antibodies. This is likely due to the relatively stringent criteria used in this study for the calculation of the cutoff and in estimating reinfections to ensure the detection of only the coronavirus species-specific antibodies. We observed that antibodies against seasonal HCoVs start to develop at an early age and the majority of children have antibodies against at least one HCoV by the age of 3 years. The participating children had no underlying diseases and thus this study provides information on the circulation of HCoVs in the general child population thereby avoiding biases caused by symptom-based sampling. Also, since the loss of maternal antibodies against seasonal HCoVs occurs by the age of 6 months (18, 19), the sera collected from children at 1 year of age (13 months) or later likely have antibodies that were developed following infections by circulating HCoVs. By the age of 1 year, 6–21% of children were seropositive against different coronavirus types, which represent numbers lower than the ones reported in other age-stratified FIG 5 Summary of seasonal coronavirus infections and reinfections in 140 children with follow-up samples at ages 1, 2 and 3 years. Status of seropositivity, infection, or reinfection for 229E, HKU1, NL63, and OC43 are based on anti-HCoV IgG antibody results in N antibody-specific enzyme immunoassay (EIA). Reinfection is determined as a significant (.20 EIA units) increase in serum anti-HCoV IgG antibody levels between the sequential serum samples. The numbers refer to the number of seropositive and reinfected children. The percentages refer to the percentage of children from the whole cohort (n= 140). Seasonal Coronavirus Seroprevalence in Children Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.01967-21 8 Downloaded from https://journals.asm.org/journal/spectrum on 10 May 2022 by 130.234.243.249.